The Structures of CuA and Cytochrome a in Cytochrome c Oxidase

Citation

Martin, Craig Timothy (1985) The Structures of CuA and Cytochrome a in Cytochrome c Oxidase. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/eft1-pp43. https://resolver.caltech.edu/CaltechTHESIS:04052019-144431487

Abstract

Cytochrome c oxidase contains four metal centers, two heme irons and two copper centers. The two low-potential centers, Cu_A and cytochrome a, function in the transfer of electrons from cytochrome c to the oxygen binding site within cytochrome oxidase. Although almost nothing is currently known about the mechanism of proton pumping in cytochrome oxidase, it is very likely that at least one of these low-potential metal centers is directly involved in the translocation of protons across the inner mitochondrial membrane. Essential to a complete understanding of the mechanisms of electron transfer and proton pumping in cytochrome oxidase is a knowledge of the structures of the involved metal centers.

In this work, the spectroscopic tool of electron nuclear double resonance (ENDOR) is used in conjunction with the direct incorporation of isotopically substituted amino acids into the yeast protein to determine the identities of ligands to Cu_A and cytochrome a in cytochrome oxidase. For cytochrome a, the incorporation of [1,3-¹⁵N₂]histidine into the yeast protein results in the appearance of an ENDOR signal associated with hyperfine coupling to the histidine ring ¹⁵N, thereby unambiguously identifying histidine as an axial ligand to cytochrome a. Comparison of this result with similar results in the native and ¹⁵N-substituted bis-imidazole model compounds metmyoglobin-imidazole and bis-imidazole tetraphenyl porphyrin, provides strong evidence that cytochrome a is coordinated by two axial histidine ligands.

Similar studies on the effect of the incorporation of [1,3-¹⁵N₂]histidine into yeast cytochrome oxidase demonstrate that histidine is a ligand to Cu_A. The incorporation of [β,β-²H₂]cysteine into the protein reveals that the two strongly coupled protons previously observed in the ENDOR spectrum of Cu_A (H. L. Van Camp, et al. (1978) Biochim. Biophys. Acta 537, 238) can be assigned to the methylene protons on cysteine, proving conclusively that cysteine is a ligand to [β-¹³C]cysteine-substituted protein. These results, combined with the resolution in the native enzyme of a new hyperfine coupling to a third strongly coupled proton for Cu_A, provide strong evidence for the coordination of two cysteine ligands to Cu_A.

Armed with the identification of one histidine and two cysteine ligands to Cu_A, we present a model for the physical and electronic structure of the oxidized Cu_A center. This model is characterized by the delocalization of a substantial amount of unpaired spin density away from the copper atom and into an orbital between the two cysteine sulfur ligands. This model satisfactorily explains many of the unusual spectroscopic features of Cu_A and should prove to be of use in future studies of the nature of the Cu_A site.

With the positive identification of ligands to cytochrome a and Cu_A, we have analyzed the available protein sequence data for the subunits of cytochrome oxidase from a wide variety of organisms, and are able to reach some conclusions regarding the physical location of these metal centers in the subunit structure of the protein. The cysteine ligand(s) to Cu_A most certainly derive from the two conserved cysteine residues in subunit II. Furthermore, it is predicted that all of the metal centers in cytochrome oxidase cannot be located in subunit II, and are quite likely shared with subunit I. Finally, we present a model for the protein coordination of Cu_A in which the two cysteine sulfur ligands to Cu_A originate in two cysteine residues which are physically adjacent on the face of an α-helix in subunit II.

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